Radiation and systemic chemotherapy are important therapeutic modalities for the treatment of cancer. Nuclear DNA is considered to be a major cellular target responsible for the cytotoxicity of ionizing radiation and many conventional antineoplastic drugs. As a consequence, the levels of DNA damage and its repair are likely to influence cell survival and affect clinical outcome (1).
The manipulation of DNA repair systems has recently become the focus of considerable interest as a means of enhancing the efficacy of radio- and chemotherapy. Particular emphasis has been placed on single and double-strand break repair pathways (2). Small molecule inhibitors have now been developed that target enzymes such as poly(ADP-ribose) polymerase (PARP) and apurinic/apyrimidinic endonuclease (APE 1), which are involved in the repair of damaged bases and single-strand breaks induced by many agents including ionizing radiation and alkylating agents (1, 3, 4); tyrosyl DNA-phosphodiesterase (Tdp1), which is required for the repair of strand breaks introduced by topoisomerase 1 inhibitors such as camptothecin and irinotecan (5); and ATM and DNA-PK, which regulate the response to DNA double-strand breaks (6, 7). Inhibitors of PARP are now in clinical trial (8).
Ionizing radiation and other genotoxic agents often generate strand breaks with incompatible termini that must be processed in order for single and double-strand break repair pathways to complete the repair. Among the frequently observed termini are 3′-phosphate and phosphoglycolate and 5′-hydroxyl groups (9, 10). These lesions create a barrier for DNA polymerases and ligases to replace missing bases and seal the breaks because these enzymes have a strict requirement for the presence of a 3′-hydroxyl group and in addition DNA ligases require a 5′-phosphate group (11, 12).
A major enzyme responsible for the phosphorylation of 5′-hydroxyl termini and dephosphorylation of 3′-phosphate termini in human cells is polynucleotide kinase/phosphatase (hPNKP) (13, 14). In the single-strand break (SSB) repair pathway hPNKP acts in concert with XRCC1, DNA polymerase β and DNA ligase III (15-17). PNKP-mediated DNA end-processing at double-strand breaks is a component of the nonhomologous end-joining (NHEJ) pathway and is dependent on DNA-PKcs and XRCC4 (18-20). In addition to its role in the repair of strand breaks produced directly by genotoxic agents, hPNKP has been implicated in the repair of strand breaks produced by enzymatic processes, including strand breaks introduced by the βδ-AP lyase activity of DNA glycosylases such as NEIL1 and NEIL2 (21, 22), which generate 3′-phosphate termini. Similarly, hPNKP is required to process termini generated by the topoisomerase I inhibitor camptothecin (23). Treatment with camptothecin stalls topoisomerase I while it is covalently attached to a 3′-phosphate group in the course of its nicking-resealing activity. The stalled enzyme can be cleaved from the DNA by Tdp1 leaving a strand break with 3′-phosphate and 5′-hydroxyl termini, which necessitates the subsequent action of PNKP. Down-regulation of hPNKP by RNAi sensitized cells to a variety of genotoxic agents including ionizing radiation, camptothecin, methyl methanesulfonate and hydrogen peroxide (24). It remains to be determined which of hPNKP's activities, 5′-kinase or 3′-phosphatase (or both), is responsible for sensitization to each agent. The two activities are independent with separate DNA binding domains (25), but the phosphatase reaction appears to proceed ahead of the kinase reaction (26).
Synthetic lethality occurs when a combination of two protein knockouts is lethal, however the corresponding single mutations are viable. The original concept of synthetic lethality as it relates to DNA repair was discovered in 2005. The Ashworth and Helleday groups published two papers back to back in Nature, outlining synthetic lethality between BRCA−/− cells and inhibition of poly(ADP-ribose) polymerase (PARP).
It is, therefore, desirable to provide inhibitors of DNA repair proteins such as polynucleotide kinase/phosphatase and poly(ADP-ribose) polymerase, and their compounds, compositions, methods and kits and uses thereof.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the present invention.